An oligonucleotide which is active in stimulating immunoresponse (hereinafter sometimes referred to as an immunostimulating oligonucleotide, immunostimulating nucleic acid or immunostimulating DNA) was discovered by T. Tokunaga and others in 1984 in the course of a search for antitumoral substances against BCG. It was then elucidated that the activity is due to a specific base sequence containing a dinucleotide of cytosine and guanine (5′-CpG-3′: the so-called CpG sequence) (Tokunaga, T., et al., J. Natl, Cancer Inst., 72, 955 (1984): Non-patent Reference 1; Tokunaga, T., et al., J. Natl. Cancer Res., 79, 682 (1988): Non-patent Reference 2).
Genome DNAs containing a CpG sequence present in organisms other than vertebrates and plants are also found to have a similar activity. It is considered that a sequence around the CpG core is also important in immunostimulation activity. Particularly, the sequence of 5′-PuPuCpGPyPy-3′, in which unmethylated CpG is sandwiched by substituted purines (Pu) and substituted pyrimidines (Py), is recognized as a typical unmethylated CpG motif (Krieg, A., et al., Nature, 374, 576 (1995): Non-patent Reference 3). As well known, a CpG motif is defined as a short nucleotide sequence (generally, a sequence of four to ten nucleotides) containing at least one cytosine (C)-guanine (G) sequence in which the 5′ position of the cytosine in the cytosine-guanine sequence is not methylated. Hereinafter, CpG is used to mean unmethylated CpG unless otherwise noted.
Examples of useful CpG motifs (hexamers) are given below, wherein A denotes adenine, G guanine, T thymine, and U uracil, respectively:
AACGTT, AGCGTT, GACGTT, GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, GGCGCC, AACGCT, AGCGCT, GACGCT, GGCGCT
An oligonucleotide of 8 to 100 nucleotides containing an above-mentioned sequence has an immunostimulation activity (Japanese Patent Application Publication No. 2001-503254: Patent Reference 1)
The following are examples of immunostimulating oligonucleotides containing a CpG motif, which have been reported as being effective in activating NK cells, wherein the underlined parts show a CpG motif and the parts with capital letters denote a thiolated DNA (Iho, S., and Yamato, S., Annual Review Immunity, 2001, 137-146(2002): Non-patent Reference 4):
accgataccggtgccggtgacggcaccacg(SEQ ID NO 7) accgatagcgctgccggtgacggcaccacg(SEQ ID NO 8) accgatgacgtcgccggtgacggcaccacg(SEQ ID NO 9) accgattcgcgagccggtgacggcaccacg(SEQ ID NO 10) ggggggggggggcgatcggggggggggggg(SEQ ID NO 11) gggggggggggacgatcgtcgggggggggg(SEQ ID NO 12) ggggggggggggaacgttgggggggggggg(SEQ ID NO 13) GAGAACGCTCGACCTTCGAT(SEQ ID NO 14) TCCATGACGTTCCTGATGCT(SEQ ID NO 15) TCTCCCAGCGTGCGCCAT(SEQ ID NO 16) GGggtcaacgttgaGGGGGg(SEQ ID NO 17)
There are known several types of sequences as immunostimulating nucleic acids other than CpG motifs. Examples include a T-rich nucleic acid such as 5′TTT3′ which is rich in thymidine, a G-rich nucleic acid such as 5′GGGG3′ which is rich in guanidine, a TG-rich nucleic acid which is rich in thymidine and guanidine, and a C-rich nucleic acid which is rich in cytidine. Recently these sequences have received considerable attention as non-CpG immunostimulating nucleic acids (Japanese Patent Application Publication No. 1996-500738: Patent Reference 2; Japanese Patent Application Publication No. 2002-512599: Patent Reference 3; Japanese Patent Application Publication No. 2003-51028: Patent Reference 4; Japanese Patent Application No. 2003-510290: Patent Reference 5).
A characteristic effect of the above-mentioned immunostimulating nucleic acids on immunocytes is that they activate antigen presenting cells. They act directly upon such cells as monocytes, macrophages or dentritic cells to produce immunoenhancing cytokines such as IL-6, TNF-α, IL-12, IFNα/β, IL-18 or nitrogen monoxide.
Recently there has been seen an increase in the number of patent applications on nucleic acids for therapeutic purposes or DNA vaccine compositions against immunological diseases. For example, the University of Iowa Research Foundation has proposed a number of sequences based on CpG motifs for use in therapies on adjuvants against diseases or disorders, including immunodeficiencies caused by infections with viruses, bacteria, fungi or parasites, cancers, or acute reduction in the air current due to exposure to lipopolysaccharides or endotoxins (Japanese Patent Application Publication No. 1998-506265: Patent Reference 6; Japanese Patent Application Publication No. 2001-503267: Patent Reference 7; Japanese Patent Application No. 2001-513776: Patent Reference 8).
A patent application is found on the use of a CpG motif in DNA vaccines for fishery products (Japanese Patent Application Publication No. 1997-285291: Patent Reference 9).
A patent application has also been filed on the use of a CpG motif for preventive purposes against infection with parvovirus in animals (Japanese Patent Application No. 2000-509976).
Aside from that set out in Patent Reference 1, a number of sequences exhibiting an immunostimulant activity are also set out in patent applications such as Patent References 11 and 12 (Japanese Patent Application Publication No. 2002-517156: Patent Reference 11; Japanese Patent Application No. 2002-526425: Patent Reference 12).
As in the case of gene therapy using an antisense DNA, an immunostimulating nucleotide is often modified so that the phosphodiester bonds at its phosphoric acid backbone are converted to phosphorothioate bonds so as to be imparted with resistance against nuclease. Besides, it is often a case that an oligonucleotide is used concurrently with a transfection agent such as a liposome, cationic lipid, or cholesterol, for the purpose of enhancing the affinity with cells.
Some retroviruses and adenoviruses provided, at the beginning, a promising prospect in vitro as a transfection agent for antisense DNAs in gene therapy. However, their uses are now very limited because of their inflammatory and immunogenic nature as well as the risk of mutagenesis and integration with the genome due to such naturally occurring viruses (Mulligan, Science, 260, 926-932 (1993): Patent Reference 5; Miller, Nature, 357, 455-460 (1992): Patent Reference 6; Crystal, Science, 270, 404-410 (1995): Patent Reference 7).
As an alternative to such natural types of transfection agents for genes, there is proposed an artificial and nonviral carrier, which is easy to handle as compared with viruses and enables assured and efficient introduction of DNAs into cells (Tomlinson and Rolland, J. Contr. Rel., 39, 357-372 (1996): Patent Reference 8).
The nonviral, artificial carrier now under extensive studies is polyethyleneimine (PEI). PEI is a cationic polymer, which assumes a three-dimensional branched structure in a variety of adherent cell or suspended cell lines, that can achieve a transfection efficiency above average in some cases (Boussif et al., Gene Therapy, 3, 1074-1080 (1996): Non-patent Reference 9).
There have been many patent applications on various types of cationic polymers or cationic lipids, modified with a substituent containing nitrogen atom as in PEI, filed under titles such as gene carrier, transfection agent, pharmaceutical support and the like.
However, the present situation is that almost no investigation has been made on the safety of cationic polymers including PEI. While the presence of amino group(s) is generally indispensable in order to render a substance cationic, the substance with amino group(s) is highly bioactive and has a risk of toxicity in the body. As a matter of fact no cationic polymers studied so far have been put into practical use or listed in dictionaries on pharmaceutical additives or the like (The Pharmaceutical Additives Dictionary, Edited by Pharmaceutical Additives Association of Japan, Published by Yakujinipposha: Non-patent Reference 11).
β-1,3-glucan is a polysaccharide which has been put into clinical use in intramuscular injection. It has been long known that this polysaccharide assumes a triple helix structure as it occurs naturally (Theresa M. McIntire and David A. Brant, J. Am. Chem. Soc., 120, 699 (1998): Non-patent Reference 12). The in vivo safety of this polysaccharide has already been confirmed since it has been actually put into practice over twenty years as a intramuscular injection in the immunological enhancement treatment against gynaecological cancer (Shimizu et al., Biotherapy, 4, 1390 (1990): Non-patent Reference 13; Hasegawa, Oncology and Chemotherapy, 8, 225 (1992): Non-patent Reference 14).
The prior art includes conjugation of β-1,3-glucan with a biomaterial such as DNA for use as a gene carrier. This prior art relates to the preparation of a conjugate of β-1,3-glucan and a bioactive material in which β-1,3-glucan of triple helix structure as it naturally occurs is bonded to the bioactive material through covalent linkage (PCT/US95/14800: Patent Reference 13).
Recently, the present inventors and others have discovered that a polysaccharide having β-1,3-bonds in the backbone forms a new type of complex with various nucleic acids when subjected to a specific artificial treatment (PCT/JP00/07875: Patent Reference 14; PCT/JP02/02228: Patent Reference 15; Sakurai, K., et al., J. Am. Chem. Soc., 122, 4520 (2000): Non-patent Reference 15; Sakurai, K., et al., Chem. Lett., 1242 (2000): Non-patent Reference 16).
The object of the present invention is to provide an immunostimulating agent in which an immunostimulating oligonucleotide is complexed with a new type of carrier which is safe and has a high transfection efficiency.